Analysis of a WRN-like mitochondrial protein (WLMP) - identity and role in mitochondrial DNA damage responses

Understanding the biology of normal human ageing is key to improving health during later life. Because ageing is a gradual, long-term process, it is very hard to study in the laboratory. However, by looking at premature ageing diseases, we can start to identify key molecules involved in ageing. The best model system we have is the premature ageing disease called Werner's syndrome. People with this syndrome first show signs of early ageing such as grey hair, wrinkled skin and age-related diseases including hardening of the arteries, cancer and diabetes in their teens or twenties, and have a much shorter than average lifespan. Scientists have identified a single region of the genetic blueprint (a gene) which when lost causes Werner's syndrome. This is particularly exciting as it is possible to work out the function of the gene, which is called WRN, in the test tube and to examine the effect of losing this gene on the ageing of human cells grown in dishes in the lab. We now know from these studies that the protein made from the WRN gene acts to cut or unwind unusual DNA structures, preventing such DNA from rearranging itself - a dangerous event that can lead to the cells ageing prematurely or even leading to cancer. Another key aspect of normal ageing is the accumulation of damage in our DNA throughout our lives, so that older people have high levels of DNA damage. Many things cause damage, but perhaps one of the most important is a dangerous form of oxygen made as a by-product of producing the energy needed for life. This energy production happens in the cell's powerhouses, called mitochondria, which have their own DNA. Because the dangerous form of oxygen is made in the mitochondria right next to mitochondrial DNA, it is widely believed that the DNA in mitochondria is very prone to damage. We have discovered that the WRN protein, or a protein that looks very similar to it, is present in the mitochondria. This is a very exciting new finding that suggests a direct link between what we know about premature ageing in people and ageing due to mitochondrial DNA damage. We want to carry out a series of experiments designed to find out whether the protein we see in mitochondria really is WRN. We shall also see whether more WRN goes into mitochondria when their DNA gets damaged, and we shall try to discover the signal that is responsible for sending WRN to damaged mitochondria. If we find a direct role for WRN in mitochondrial DNA repair, this will open up a whole new research field that may in the longer term help in the development of drugs to slow down mitochondrial DNA damage, prevent premature ageing and enhance the quality of life of older people.

Werner's syndrome (WS) is the best current model of human ageing, and is caused by mutation of WRN, which encodes a helicase/exonuclease. WRN resolves aberrant DNA structures arising during DNA replication, suppressing illegitimate recombination. Whilst WS is useful in analysing cellular senescence, it has not until now been possible to link cellular senescence with the mitochondrial theory of ageing. Accumulation of ROS-induced DNA damage within mitochondria is thought to be a major contributor to ageing, but the mechanisms by which the cell deals with mtDNA damage are at best unclear. Our novel and exciting preliminary data suggest localisation of WRN to mitochondria: the presence of WRN in organelles whose DNA is subject to high levels of damage is suggestive that WRN may play a role in mtDNA repair. Our data are based upon immunofluorescence (IF) and immunoblotting (IB) studies, but we cannot yet be sure that the protein detected is genuinely WRN. We therefore term the protein WRN-like mitochondrial protein, or WLMP. In this one year 'proof of principle' project, we shall establish the identity of WLMP. Using a panel of anti-WRN antibodies, we shall confirm our IF/IB data, then sequence immunoprecipitated WLMP from isolated mitochondria, and in parallel, generate peptide fingerprints by mass spectroscopic analysis. If WLMP is identified as WRN, we shall attempt to localise a mitochondrial targeting sequence by generating a series of GFP-tagged WRN deletions and assaying mitochondrial uptake. The role of the electrochemical gradient in WLMP accumulation will be assessed using uncoupling agents such as rotenone. To analyse a possible role of WLMP in mtDNA repair, we shall perform co-localisation studies of WLMP with mtDNA by confocal and electron microscopy. WLMP association with mtDNA will be determined in cells with endogenous DNA damage (mutant for POLG, twinkle helicase or p53), or those damaged by ddC and other drug treatments.